HIV-1 is responsible for an ongoing pandemic that has affected tens of millions of people, and other retroviruses cause both human and animal diseases. A better understanding of the fundamental principles of retrovirus replication should serve as an underpinning for the development of additional Pharmaceuticals and eventually of immunological intervention. A critical step in the life cycle of a retrovirus is assembly of the virus particle from viral proteins, the viral RNA genome, and the lipid membrane from the infected host cell. The focus of this proposal is to unravel several aspects of retrovirus assembly, using mainly the tractable and well-studied avian Rous sarcoma virus as a model system, but including some focused experiments on HIV-1 proteins. Major Specific Aim 1 is to characterize the interaction of the RSV and HIV-1 internal structural protein, Gag, with membranes. The experiments will address: (1a) the origin of the raft-like viral membrane in RSV and HIV-1, (1b) the role of phosphatidyl inositol bisphosphate (PIP2) in membrane targeting of HIV-1 Gag, and (1c) the in vivo functions of the membrane-interacting domain (MA) of RSV Gag. Major Specific Aim 2 is to characterize the protein-protein interactions and protein-RNA interactions in assembly of an immature virus particle. The experiments will address: (2a) the structural function of a short sequence of amino acids that promotes immature assembly in RSV Gag, (2b) the mechanism by which the RNA binding domain of RSV Gag, NC, promotes protein dimerization that then leads to assembly, (2c) the role of inositol hexakisphosphate (IP6) in promoting proper assembly of HIV-1 Gag in vitro, and (2d) the nature of the Gag-RNA interactions leading to specific packaging of the RSV genome. Much of the proposed work is grounded in biochemical approaches, in particular relying on an in vitro assembly system developed in our lab.